MKK7 activation inhibitor

ABSTRACT

PAK4 and JIK, both of which bind to MKK7 and directly phosphorylate MKK7, were found in the present invention. The present invention provides an inhibitor of c-Jun phosphorylation caused by JNK3 and a method for inhibiting the same, and an agent for preventing and/or treating a disorder attributable to c-Jun phosphorylation caused by JNK3 and a method for preventing and/or treating the same, all of which comprise inhibiting one member selected from the following: the binding of PAK4 to MKK7, the phosphorylation of MKK7 by PAK4, the binding of JIK to MKK7, and the phosphorylation of MKK7 by JIK. Further, the present invention provides a method for identifying a compound that inhibits the binding of PAK4 to MKK7, the phosphorylation of MKK7 caused by PAK4, the binding of JIK to MKK7, or the phosphorylation of MKK7 caused by JIK, as well as the compound obtained thereby. Furthermore, the present invention provides a pharmaceutical composition containing an effective amount of at least one member selected from the group consisting of the aforementioned compound and the aforementioned inhibitor.

TECHNICAL FIELD

The present invention relates to the inhibition of c-Jun phosphorylationcaused by c-Jun N-terminal kinase 3 (JNK3), comprising inhibiting theactivation of MAP kinase kinase 7 (MKK7), and to the improvement of adisorder attributable to c-Jun phosphorylation by JNK3, as well as toimprovement of a neurodegenerative disorder. More particularly, thepresent invention relates to an inhibitor of c-Jun phosphorylationcaused by JNK3 and a method for inhibiting the same, comprisinginhibiting the interaction of MKK7 with p21-activated kinase 4 (PAK4)and/or the interaction of MKK7 with JNK/SAPK-inhibitory kinase (JIK),that is, inhibiting the MKK7 activation that results from the binding ofPAK4 to MKK7 followed by direct phosphorylation of MKK7 by PAK4 and/orthat results from the binding of JIK to MKK7 followed by directphosphorylation of MKK7 by JIK. The present invention relates further toa preventive agent and/or an inhibitor of a disorder attributable toc-Jun phosphorylation by JNK3, and a method for preventing and/orinhibiting the same, as well as to a preventive agent and/or aninhibitor of neurodegeneration, and a method for preventing and/orinhibiting the same, all of which has the aforementioned features. Thepresent invention relates still further to a method for identifying acompound that inhibits the binding of PAK4 to MKK7, the phosphorylationof MKK7 by PAK4, the binding of JIK to MKK7, or the phosphorylation ofMKK7 by JIK, and to a compound that is obtained by the identificationmethod.

BACKGROUND ART

c-Jun N-terminal kinase (hereinafter abbreviated as JNK) is a proteinkinase that belongs to the MAP kinase (hereinafter abbreviated as MAPK)family. Three JNK genes (JNK1, JNK2 and JNK3) have been discovered inmammals. Among these, JNK3 is selectively expressed, for example, in thebrain-nervous system.

Unlike the classical MAPK, JNK3 is barely activated by proliferationstimuli. The activation of JNK3 is caused by stress on a cell (e.g., DNAdamage, ultraviolet radiation, employing an assay system using a signaland/or a marker that makes it possible to detect the binding of PAK4 toMKK7 or the phosphorylation of MKK7 by PAK4, and detecting the presenceor the absence of the change of the signal and/or the marker.

The identification method using cells as described above can be used incombination with the in-vitro identification methods described above.Compounds that inhibit the binding of PAK4 to MKK7 and/or thephosphorylation of MKK7 caused by PAK4, which are obtained by thein-vitro identification methods as described above, can be subjected tofurther experimentation with the identification methods using cells asdescribed above, in order to select useful compounds.

When JIK is used in place of PAK4 in the aforementioned identificationmethod, a compound that inhibits the binding of JIK to MKK7 or thephosphorylation of MKK7 caused by JIK can be also identified.

The identification method according to the present invention providescompounds that inhibit the binding of PAK4 to MKK7, compounds thatinhibit the binding of JIK to MKK7, compounds that inhibit thephosphorylation of MKK7 caused by PAK4 and compounds that inhibit thephosphorylation of MKK7 caused by JIK. These compounds are also includedin the scope of the present invention. These compounds are available asinhibitors of the binding of PAK4 to MKK7, inhibitors of thephosphorylation of MKK7 by PAK4, inhibitors of the binding of JIK toMKK7 and inhibitors of the phosphorylation of MKK7 by JIK. Suchcompounds can be exemplified by peptides or oligopeptides, comprisingthe amino acid sequences of the interaction sites such as, for example,the binding sites of the two proteins. Such peptides or oligopeptidescan be identified by designing them from the amino acid sequence of PAK4or MKK7, synthesizing them by peptide synthesis methods well-known inthe art, and examining, in the identification method described above,whether they can inhibit the binding of PAK4 to MKK7, thephosphorylation of MKK7 caused by PAK4, the binding of JIK to MKK7 orthe phosphorylation of MKK7 caused by JIK. Examples of theaforementioned compounds also include antibodies capable of inhibitingthe binding of PAK4 to MKK7 or the binding of JIK to MKK7. Suchantibodies can be obtained by preparing the antibodies against PAK4, JIKor MKK7 and selecting the one capable of heat, high osmotic pressure,endoplasmic reticulum stress, active oxygen) or by an inflammatorycytokine (e.g., tumor necrosis factor (TNF), interleukin-1 (1L-1)). Theactivated JNK is understood to translocate from the cytoplasm into thenucleus and control the expression of target genes via thephosphorylation of transcription factors, such as c-Jun.

The activation of JNK is involved in apoptosis induced by various stressstimuli. For example, it has been reported that JNK is activated innerve cell death resulting from withdrawal of nerve growth factor (NGF,non-patent document 1), and that nerve cell death resulting fromwithdrawal of NGF is suppressed by expression of a dominant negativec-Jun mutant (non-patent document 2). In addition, it has been reportedthat excitatory neuron death induced by administration of kainic acid issuppressed in a JNK3 knock-out mouse (non-patent document 3). The abovefindings suggest that the activation of JNK3 is involved in nerve celldeath.

MKK4 and MKK7, which are members of the MAPK kinase family (hereinafterabbreviated as MAPKK), are known to activate JNK. MKK7 is also calledMAPKK7, MAP2K7 and JNKK2, and specifically phosphorylates and activatesJNK (non-patent document 4 and 5). In contrast, MKK4 phosphorylates andactivates JNK as well as phosphorylates and activates ERK2 and p38,which are also members of MAPK family. Since JNK activation by osmoticpressure stimulus or ultraviolet radiation is still observed in anembryonic stem cell (ES cell) with a disrupted MKK4 gene (non-patentdocument 6), MKK7 is considered to work on the activation of JNKindependently of MKK4.

The activation of JNK is also caused by a signal from cdc42, which isone of the low-molecular weight GTP proteins (non-patent document 7).PAK (p21-activated kinase) is known to be a kinase that binds to cdc42and transmits a signal from cdc42. Actually, it has been reported thatthe JNK signaling pathway is activated by over-expression of PAK1, PAK2,PAK3 or PAK4, all of which are members of the PAK family (non-patentdocument 8, 9, 7 and 10, respectively). However, the detailed mechanismof the signaling pathway between PAK and the JNK activation has not yetbecome clear. For example, whether the signaling is direct or indirectis not clear.

There have been some reports suggesting the involvement of cdc42 innerve cell death. For example, forced expression of activated cdc42 innerve cells induces nerve cell death, whereas a dominant negative cdc42mutant suppresses nerve cell death resulting from withdrawal of NGF(non-patent document 11). In addition, it is reported that activatedcdc42 activates MKK7 as well as activates JNK (non-patent document 12).Therefore, the signaling pathway from cdc42 to JNK, which is mediated byMKK7, may be possibly involved in nerve cell death.

Endoplasmic reticulum stress (hereinafter abbreviated as ER stress) isamong the stresses causing the activation of JNK3. ER stress is causedby the accumulation of abnormal proteins in the endoplasmic reticulum(hereinafter may be abbreviated as ER) as a result of a defect in theprotein folding process in the ER due to various stimuli (e.g., glucoseexhaustion, change in homeostasis of calcium concentration, activeenzyme). When ER stress occurs, expression of endoplasmic reticulummolecular chaperon is induced (that is, unfolded protein response: UPR),thus eliminating the misfolding. IRE1 is known to work in this processas an ER stress sensor protein (non-patent document 13).

It has been reported that IRE1 and TRAF2 are involved in the process ofJNK activation caused by ER stress (non-patent document 14 and 15).Concretely, the IRE1 disrupted cell line shows suppressed JNK activationin response to ER stress, whereas over-expression of IRE1 activates JNK.In addition, IRE1 binds to TRAF2, and a dominant negative TRAF2 mutantsuppresses the JNK activation by IRE1.

In addition to the above, JIK (also referred to as DPK) is also known asa protein that is involved in the process of JNK activation by ERstress. It is considered that JIK binds to IRE1 and TRAF2, and isinvolved in the JNK activation by ER stress. For example,over-expression of JIK augments the JNK activation by ER stress, whereasan active-site-deletion mutant of JIK suppresses the JNK activation byER stress (non-patent document 15).

JIK is one of the STE20-related serine/threonine kinases that are humanhomologs of yeast Ste20p protein. In addition to the aforementionedactions of JIK, it has been reported that JIK inhibits the JNKactivation by epidermal growth factor (EGF) stimulus, while the activityof JIK itself is suppressed (non-patent document 16), and that theover-expression of JIK leads to the JNK activation (non-patent document17).

On the other hand, overload of ER stress is known to induce apoptosis.Since ischemia or accumulation of an abnormal protein such aspolyglutamine or amyloid β (hereinafter abbreviated as Aβ) possiblygives rise to ER stress, it is pointed out that there is a relationshipbetween nerve cell death due to ER stress and a neurodegenerativedisorder.

Since apoptosis induced by ER stress was suppressed by each dominantnegative mutant of MKK4 and MKK7 (non-patent document 18), JNKactivation via MKK4 or MKK7 is likely to be involved in the apoptosisinduced by ER stress.

The literature cited in the present specification is listed below.

Patent document 1: International Patent Publication WO 01/67299.

Non-patent document 1: Eilers A. et al., J. Neurosci., 1998, Vol. 18,pp. 1713-1724.

Non-patent document 2: Ham J. et al., Neuron, 1995, Vol. 14, pp.927-939.

Non-patent document 3: Yang D. et al., Nature, 1997, Vol. 389, pp.865-870.

Non-patent document 4: Moriguchi T. et al., EMBO J., 1997, Vol. 16, pp.7045-7053.

Non-patent document 5: Foltz I. et al., J. Biol. Chem., 1998, Vol. 273,pp. 9344-9351.

Non-patent document 6: Yang D. et al., Proc. Natl. Acad. Sci. U.S.A.,1997, Vol. 94, pp. 3004-3009.

Non-patent document 7: Bagrodia S. et al., J. Biol. Chem., 1995, Vol.270, pp. 27995-27998.

Non-patent document 8: Brown J. et al., Curr. Biol., 1996, Vol. 6, pp.598-605.

Non-patent document 9: Frost J. et al., Mol. Cell. Biol., 1996, Vol. 16,pp. 3707-3713.

Non-patent document 10: Abo A. et al., EMBO J., 1998, Vol. 17, pp.6527-6540.

Non-patent document 11: Bazenet C. et al., Proc. Natl. Acad. Sci.U.S.A., 1998, Vol. 95, pp. 3984-3989.

Non-patent document 12: Foltz I. et al., J. Biol. Chem., 1998, Vol. 273,pp. 9344-9351.

Non-patent document 13: Urano F. et al., J. Cell Sci., 2000, Vol. 113,pp. 3697-3702.

Non-patent document 14: Urano F. et al., Science, 2000, Vol. 287, pp.664-666.

Non-patent document 15: Yoneda T. et al., J. Biol. Chem., 2001, Vol.276, pp. 13935-13940.

Non-patent document 16: Tassi E. et al., J. Biol. Chem., 1999, Vol. 274,pp. 33287-33295.

Non-patent document 17: Zhang W. et al., Biochem. Biophys. Res. Commun.,2000, Vol. 274, pp. 872-879.

Non-patent document 18: Zhang C. et al., Biochem. Biophys. Res. Commun.,2001, Vol. 289, pp. 718-724.

Non-patent document 19: Cell Technology, 2001, Vol. 20, No. 11, FeatureStory: The mechanism of onset of neurodegenerative disorders and theperspective of treatment thereof.

DISCLOSURE OF THE INVENTION

In view of the current situation described above, to inhibit any one ofthe stages in the mechanism of JNK activation by various stresses makesit possible to more clearly understand a disorder attributable toapoptosis induced by the JNK activation, such as, more specifically, aneurodegenerative disorder, as well as to prevent and/or treat such adisorder. For example, discovery of a protein capable of interactingwith MKK7 and inhibiting the MKK7 activation would make it possible toinhibit the JNK activation.

The present inventors made an in-silico prediction of the interaction ofMKK7 with PAK4 or JIK, and proved the interaction experimentally, andfurther discovered that the interaction leads to the phosphorylation ofMKK7 by PAK4 or JIK followed by the activation of the JNK3 signalingpathway, all of which discoveries contribute to the achievement of thepresent invention.

More specifically, the first aspect of the present invention relates toan inhibitor of c-Jun phosphorylation by c-Jun N-terminal kinase 3,having at least one function selected from the group consisting of thefollowing functions:

i) inhibiting the binding of p21-activated kinase 4 (PAK4) to MAP kinasekinase 7 (MKK7);

ii) inhibiting the phosphorylation of MKK7 caused by PAK4;

iii) inhibiting the binding of JNK/SAPK-inhibitory kinase (JIK) to MAPkinase kinase 7 (MKK7); and

iv) inhibiting the phosphorylation of MKK7 caused by JIK.

Another aspect of the present invention relates to a method forinhibiting c-Jun phosphorylation caused by c-Jun N-terminal kinase 3,comprising at least one step selected from the group consisting of thefollowing steps:

i) inhibiting the binding of p21-activated kinase 4 (PAK4) to MAP kinasekinase 7 (MKK7);

ii) inhibiting the phosphorylation of MKK7 caused by PAK4;

iii) inhibiting the binding of JNK/SAPK-inhibitory kinase (JIK) to MAPkinase kinase 7 (MKK7); and

iv) inhibiting the phosphorylation of MKK7 caused by JIK.

A further aspect of the present invention relates to an agent forpreventing and/or treating a disorder attributable to c-Junphosphorylation caused by c-Jun N-terminal kinase 3, having at least onefunction selected from the group consisting of the following functions:

i) inhibiting the binding of p21-activated kinase 4 (PAK4) to MAP kinasekinase 7 (MKK7);

ii) inhibiting the phosphorylation of MKK7 caused by PAK4;

iii) inhibiting the binding of JNK/SAPK-inhibitory kinase (JIK) to MAPkinase kinase 7 (MKK7); and

iv) inhibiting the phosphorylation of MKK7 caused by JIK.

A still further aspect of the present invention relates to an agent forpreventing and/or treating a neurodegenerative disorder, having at leastone function selected from the group consisting of the followingfunctions:

i) inhibiting the binding of p21-activated kinase 4 (PAK4) to MAP kinasekinase 7 (MKK7);

ii) inhibiting the phosphorylation of MKK7 caused by PAK4;

iii) inhibiting the binding of JNK/SAPK-inhibitory kinase (JIK) to MAPkinase kinase 7 (MKK7); and

iv) inhibiting the phosphorylation of MKK7 caused by JIK.

A further aspect of the present invention relates to a method forpreventing and/or treating a disorder attributable to c-Junphosphorylation caused by c-Jun N-terminal kinase 3, comprising at leastone step selected from the group consisting of the following steps:

i) inhibiting the binding of p21-activated kinase 4 (PAK4) to MAP kinasekinase 7 (MKK7);

ii) inhibiting the phosphorylation of MKK7 caused by PAK4;

iii) inhibiting the binding of JNK/SAPK-inhibitory kinase (JIK) to MAPkinase kinase 7 (MKK7); and

iv) inhibiting the phosphorylation of MKK7 caused by JIK.

A still further aspect of the present invention relates to a method forpreventing and/or treating a neurodegenerative disorder, comprising atleast one step selected from the group consisting of the followingsteps:

i) inhibiting the binding of p21-activated kinase 4 (PAK4) to MAP kinasekinase 7 (MKK7);

ii) inhibiting the phosphorylation of MKK7 caused by PAK4;

iii) inhibiting the binding of JNK/SAPK-inhibitory kinase (JIK) to MAPkinase kinase 7 (MKK7); and

iv) inhibiting the phosphorylation of MKK7 caused by JIK.

A further aspect of the present invention relates to a method foridentifying a compound that inhibits the binding of p21-activated kinase4 (PAK4) to MAP kinase kinase 7 (MKK7), comprising contacting PAK4and/or MKK7 with a test compound under conditions that allow the bindingof PAK4 to MKK7; and determining whether the test compound inhibits thebinding of PAK4 to MKK7, by detecting the presence, absence or change ofa signal generated by the binding of PAK4 to MKK7.

A still further aspect of the present invention relates to a method foridentifying a compound that inhibits the binding of JNK/SAPK-inhibitorykinase (JIK) to MAP kinase kinase 7 (MKK7), comprising contacting JIKand/or MKK7 with a test compound under conditions that allow the bindingof JIK to MKK7; and determining whether the test compound inhibits thebinding of JIK to MKK7, by detecting the presence, absence or change ofa signal generated by the binding of JIK to MKK7.

A further aspect of the present invention relates to a method foridentifying a compound that inhibits the phosphorylation of MAP kinasekinase 7 (MKK7) caused by p21-activated kinase 4 (PAK4), comprisingcontacting PAK4 and/or MKK7 with a test compound; and determiningwhether the test compound inhibits the phosphorylation of MKK7 caused byPAK4, by introducing a system using a signal and/or a marker capable ofdetecting the phosphorylation of MKK7 and detecting the presence,absence or change of the signal and/or the marker.

A still further aspect of the present invention relates to a method foridentifying a compound that inhibits the phosphorylation of MAP kinasekinase 7 (MKK7) caused by JNK/SAPK-inhibitory kinase (JIK), comprisingcontacting JIK and/or MKK7 with a test compound; and determining whetherthe test compound inhibits the phosphorylation of MKK7 caused by JIK, byintroducing a system using a signal and/or a marker capable of detectingthe phosphorylation of MKK7 and detecting the presence, absence orchange of the signal and/or the marker.

A further aspect of the present invention relates to a compound obtainedby any one of the aforementioned identification methods.

A still further aspect of the present invention relates to a compoundthat inhibits the binding of p21-activated kinase 4 (PAK4) to MAP kinasekinase 7 (MKK7).

A further aspect of the present invention relates to a compound thatinhibits the binding of JNK/SAPK-inhibitory kinase (JIK) to MAP kinasekinase 7 (MKK7).

A still further aspect of the present invention relates to a compoundthat inhibits the phosphorylation of MAP kinase kinase 7 (MKK7) causedby p21-activated kinase 4 (PAK4).

A further aspect of the present invention relates to a compound thatinhibits the phosphorylation of MAP kinase kinase 7 (MKK7) caused byJNK/SAPK-inhibitory kinase (JIK).

A still further aspect of the present invention relates to an inhibitorof the binding of p21-activated kinase 4 (PAK4) to MAP kinase kinase 7(MKK7).

A further aspect of the present invention relates to an inhibitor of thebinding of JNK/SAPK-inhibitory kinase (JIK) to MAP kinase kinase 7(MKK7).

A still further aspect of the present invention relates to an inhibitorof the phosphorylation of MAP kinase kinase 7 (MKK7) by p21-activatedkinase 4 (PAK4).

A further aspect of the present invention relates to an inhibitor of thephosphorylation of MAP kinase kinase 7 (MKK7) by JNK/SAPK-inhibitorykinase (JIK).

A still further aspect of the present invention relates to apharmaceutical composition containing an effective amount of at leastone member selected from the group consisting of the aforementionedcompounds and the aforementioned inhibitors.

A further aspect of the present invention relates to an agent forpreventing and/or treating a disorder attributable to c-Junphosphorylation caused by c-Jun N-terminal kinase 3, containing aneffective amount of at least one member selected from the groupconsisting of the aforementioned compounds and the aforementionedinhibitors.

A still further aspect of the present invention relates to an agent forpreventing and/or treating a neurodegenerative disorder, the agentcontaining an effective amount of at least one member selected from thegroup consisting of the aforementioned compounds and the aforementionedinhibitors.

A further aspect of the present invention relates to the aforementionedagent for preventing and/or treating a neurodegenerative disorder,wherein the neurodegenerative disorder is a polyglutamine disease,Huntington's disease, spino-cerebellar ataxia, bulbo-spinal muscularatrophy, dentatorubral-pallidoluysian atrophy, Alzheimer's disease, Downsyndrome, Parkinson's disease, dementia with Lewy bodies, multisystematrophy, familial amyotrophic lateral sclerosis, progressivesupranuclear palsy, corticobasal degeneration, Pick's disease, familialBritish dementia, Creutzfeldt-Jakob disease, Gerstmann-Stransslersyndrome, mad cow disease (bovine spongiform encephalopathy) (BSE), orfamilial dementia associated with neuroserpin inclusion bodies.

A still further aspect of the present invention relates to a method forpreventing and/or treating a disorder attributable to c-Junphosphorylation caused by c-Jun N-terminal kinase 3, comprising using atleast one member selected from the group consisting of theaforementioned compounds and the aforementioned inhibitors.

A further aspect of the present invention relates to a method forpreventing and/or treating a neurodegenerative disorder, comprisingusing at least one member selected from the group consisting of theaforementioned compounds and the aforementioned inhibitors.

A still further aspect of the present invention relates to theaforementioned method for preventing and/or treating a neurodegenerativedisorder, wherein the neurodegenerative disorder is a polyglutaminedisease, Huntington's disease, spino-cerebellar ataxia, bulbo-spinalmuscular atrophy, dentatorubral-pallidoluysian atrophy, Alzheimer'sdisease, Down syndrome, Parkinson's disease, dementia with Lewy bodies,multisystem atrophy, familial amyotrophic lateral sclerosis, progressivesupranuclear palsy, corticobasal degeneration, Pick's disease, familialBritish dementia, Creutzfeldt-Jakob disease, Gerstmann-Stransslersyndrome, mad cow disease (bovine spongiform encephalopathy) (BSE), orfamilial dementia associated with neuroserpin inclusion bodies.

A further aspect of the present invention relates to a reagent kitcontaining at least one member selected from the group consisting ofp21-activated kinase 4 (PAK4), JNK/SAPK-inhibitory kinase (JIK), apolynucleotide encoding PAK4, a polynucleotide encoding JIK, a vectorcontaining a polynucleotide encoding PAK4 and a vector containing apolynucleotide encoding JIK; and at least one member selected from thegroup consisting of MAP kinase kinase 7 (MKK7), a polynucleotideencoding MKK7 and a vector containing a polynucleotide encoding MKK7.

A still further aspect of the present invention relates to a reagent kitthat is used in the aforementioned identification method, containing atleast one member selected from the group consisting of p21-activatedkinase 4 (PAK4), JNK/SAPK-inhibitory kinase (JIK), a polynucleotideencoding PAK4, a polynucleotide encoding JIK, a vector containing apolynucleotide encoding PAK4 and a vector containing a polynucleotideencoding JIK; and at least one member selected from the group consistingof MAP kinase kinase 7 (MKK7), a polynucleotide encoding MKK7 and avector containing a polynucleotide encoding MKK7.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the results of an in-silico prediction of theinteraction of MKK7 with PAK4. Local alignment between MKK7 and PAK4 wasconducted, and regions with high scores are shown. The upper and lowerrows indicate partial sequences present in MKK7 and those in PAK4,respectively.

FIG. 2 shows that MKK7 was phosphorylated in-vitro by PAK4. GST-MKK7 wasphosphorylated in the presence of FLAG-PAK4 (lane 4), but notphosphorylated in the absence of FLAG-PAK4 (lane 3). On the other hand,GST was not phosphorylated either in the presence (lane 2) or theabsence (lane 1) of FLAG-PAK4. The values shown on the left-hand side ofthe figure represent molecular weights.

FIG. 3 shows that the binding of PAK4 to MKK7 was observed in a cell.The bottom panel of the figure shows the result of animmunoprecipitation test (IP), indicating that an immunoprecipitatecontaining HA-MKK7 and FLAG-PAK4 was detected in a cell lysate preparedfrom cells co-expressing HA-MKK7 and FLAG-PAK4 (lane 2), though such animmunoprecipitate was not detected in a cell lysate prepared from cellsexpressing only HA-MKK7 (lane 1). The top and middle panels,respectively, show the results of verification of the expression ofFLAG-PAK4 and HA-MKK7 in each cell lysate. Detection of theimmunoprecipitate and verification of the expression were both carriedout by western blotting (WB).

FIG. 4 shows that the temporary expression of PAK4 increased c-Junphosphorylation by JNK3 dependently on the amount of expressed PAK4. Thebottom panel in the figure shows the result of a kinase assay,indicating that GST-c-Jun(1-79) was phosphorylated when using a celllysate prepared from cells co-expressing HA-PAK4 and FLAG-JNK3 (lanes2-4), but not phosphorylated when using a cell lysate prepared fromcells expressing only FLAG-JNK3 (lanes 1). Lanes 2, 3 and 4 show theresults when transfecting with an HA-PAK4 expression vector(pcDNA-HA-PAK4) in amounts of 0.1 μg, 0.5 μg and 2.0 μg, respectively.The top and middle panels show respectively the results of verificationof the expression of FLAG-JNK3 and HA-PAK4. The verification ofexpression was carried out by western blotting (WB).

FIG. 5 illustrates the results of an in-silico prediction of theinteraction of MKK7 with JIK. Local alignment between MKK7 and JIK wasconducted, and regions with high scores are shown. The upper and lowerrows indicate partial sequences present in MKK7 and those in JIK,respectively.

FIG. 6 shows that MKK7 was phosphorylated in-vitro by JIK. GST-MKK7 wasphosphorylated in the presence of HA-JIK (lane 2), but notphosphorylated in the absence of HA-JIK (lane 1). On the other hand, GSTwas not phosphorylated in the presence (lane 3) of HA-JIK. The valuesshown on the left-hand side of the figure represent molecular weights.

FIG. 7 shows that the binding of JIK to MKK7 was observed in a cell. Thebottom panel of the figure shows the result of immunoprecipitation test(IP), indicating that an immunoprecipitate containing FLAG-MKK7 andHA-JIK was detected in a cell iysate prepared from cells co-expressingFLAG-MKK7 and HA-JIK (lane 2), though such an immunoprecipitate was notdetected in a cell lysate prepared from cells expressing only FLAG-MKK7(lane 1). The top and middle panels, respectively, show the results ofthe verification of expression of HA-JIK and FLAG-MKK7 in each celllysate. Detection of the immunoprecipitate and verification of theexpression were both carried out by western blotting (WB).

FIG. 8 shows that the temporary expression of JIK increased c-Junphosphorylation caused by JNK3. GST-c-Jun(1-79) was phosphorylated whenusing a cell lysate prepared from cells co-expressing HA-JIK andFLAG-JNK3 (lanes 3), but not phosphorylated when using a cell lysateprepared from cells expressing neither HA-JIK nor FLAG-JNK3 (lane 1) orcells expressing only FLAG-JNK3 (lanes 2).

DETAILED DESCRIPTION OF THE INVENTION

The present invention claims priority from Japanese Patent ApplicationNos. 2002-190909 and 2002-190910, which are incorporated herein byreference.

Technical and scientific terms used in the present specification havethe meanings that are normally understood by those skilled in the art,unless otherwise defined. In the present specification, reference ismade to a variety of methods known to those skilled in the art.Publications and other materials disclosing such cited known methods aredeemed completely incorporated herein in their entirety by reference.Hereinafter, a mode of embodiment of the present invention may bedescribed in more detail. The following detailed description isillustrative and merely explanatory, and does not limit the presentinvention in any way.

In the present invention, prediction of proteins that have a function tointeract with MKK7 was conducted according to the method set forth inInternational Patent Publication WO 01/67299; as a result, two proteinswere discovered. These proteins are p21-activated kinase 4 (hereinafterabbreviated as PAK4), and JIK, which is one of the STE20-relatedserine/threonine kinases. Furthermore, it was discovered by experiment,for the first time, that both PAK4 and JIK bind to MKK7 and directlyphosphorylate MKK7. In addition, it was revealed that the expression ofeach of PAK4 and JIK activates c-Jun N-terminal kinase 3 (hereinafterabbreviated as JNK3), which results in the phosphorylation of c-Jun.These findings made it clear that the JNK3 signaling pathway wasactivated as a result of the direct phosphorylation of MKK7 by each ofPAK4 and JIK.

In the present specification, “a protein having a function ofinteracting with MKK7” refers to a protein that interacts specificallywith MKK7, practically, for example, a protein that binds specificallyto MKK7 as one of its functions. More practically, “a protein having afunction of interacting with MKK7” refers to a protein capable ofphosphorylating MKK7 as one of its functions. In the presentspecification, amino acids may be represented by one letter or threeletters.

To date, it is known that PAK4 is activated by cdc42, alow-molecular-weight GTP protein (non-patent document 11). On the otherhand, it is reported that the activated cdc42 induces nerve cell deathand that the activated cdc42 or PAK4 activates the JNK signaling pathway(non-patent document 7 and 10).

We postulated, from the findings in the present invention in addition tothe information shown in those reports, the existence of a signalingpathway that comprises the activation of PAK4 by cdc42, then the bindingof activated PAK4 to MKK7 followed by the direct phosphorylation andactivation of MKK7, followed by the activation of JNK3, which leads tothe phosphorylation of c-Jun, which eventually results in the expressionof a certain physiological function. The physiological function can beexemplified by the induction of apoptosis, more practically, theinduction of nerve cell death. This finding indicates that it ispossible to inhibit the phosphorylation of c-Jun resulting from theactivation of JNK3 by inhibiting the binding of PAK4 to MKK7 and/or thephosphorylation of MKK7 caused by PAK4, and further to inhibit nervecell death. In addition, it can be possible to prevent and/or treat adisorder that is caused by the activation of the JNK3 signaling pathway,including those attributable to the cdc42-mediated activation of theJNK3 signaling pathway.

It is understood that JIK is involved in the activation of JNK caused byER stress. For example, it is reported that the over-expression of JIKaugments the activation of JNK caused by ER stress and that anactive-site-deletion mutant of JIK suppresses the activation of JNKcaused by ER stress (non-patent document 15).

We postulated, from the findings in the present invention in addition tothe information shown in those reports, that the expression of aphysiological function caused by ER stress should be mediated by asignaling pathway that comprises the activation of JIK caused by ERstress, then the binding of activated JIK to MKK7, followed by directphosphorylation and activation of MKK7, which finally results in theactivation of JNK3 that leads to the phosphorylation of c-Jun. Thephysiological function can be exemplified by the induction of apoptosis,more practically, the induction of nerve cell death. This findingindicates that it is possible to inhibit the phosphorylation of c-Junresulting from the activation of JNK3 by inhibiting the binding of JIKto MKK7 and/or the phosphorylation of MKK7 caused by JIK, and further toinhibit nerve cell death. In addition, it is possible to prevent and/ortreat a disorder that is caused by the activation of the JNK3 signalingpathway, such as a disorder attributable to the cdc42-mediatedactivation of the JNK3 signaling pathway.

A disorder that is caused by the activation of the JNK3 signalingpathway can be exemplified by a disorder attributable to apoptosis, morepractically, a neurodegenerative disorder and the like. Examples of aneurodegenerative disorder include polyglutamine diseases (e.g.,Huntington's disease, spino-cerebellar ataxia, bulbo-spinal muscularatrophy and dentatorubral-pallidoluysian atrophy), Alzheimer's disease,Down syndrome, Parkinson's disease, dementia with Lewy bodies,multisystem atrophy, familial amyotrophic lateral sclerosis, progressivesupranuclear palsy, corticobasal degeneration, Pick's disease, familialBritish dementia, Creutzfeldt-Jakob disease, Gerstmann-Stransslersyndrome, mad cow disease (bovine spongiform encephalopathy) (BSE), andfamilial dementia with neuroserpin inclusion bodies (non-patent document19), but are not limited thereto. In addition to the above examples,nerve cell death caused by ischemia or reperfusion resulting from ERstress can be prevented and/or treated.

The present invention can provide an inhibitor of c-Jun phosphorylationcaused by JNK3, a method for inhibiting c-Jun phosphorylation caused byJNK3, and further an agent for preventing and/or treating a disorderattributable to c-Jun phosphorylation caused by JNK3, such as aneurodegenerative disorder, and a method for preventing and/or treatingthe same, all of which comprises at least one feature selected from thefollowing: i) inhibiting the binding of PAK4 to MKK7; ii) inhibiting thephosphorylation of MKK7 caused by PAK4; iii) inhibiting the binding ofJIK to MKK7; and iv) inhibiting the phosphorylation of MKK7 caused byJIK.

The present invention provides, based on the aforementioned findings, amethod for identifying a compound that inhibits the binding of PAK4 toMKK7, and/or the phosphorylation of MKK7 caused by PAK4, or a compoundthat inhibits the binding of JIK to MKK7 and/or the phosphorylation ofMKK7 caused by JIK. The identification method can be established usingpharmaceutical screening systems that are well-known in the art. PAK4,JIK and MKK7 used for identifying the compound can be contained in cellsin which these are expressed by means of genetic engineering techniques,the products of cell-free synthesis systems, chemical synthesisproducts, or can be those obtained from cells or from any biologicalsamples. These can be subsequently further purified for use.Furthermore, as long as the interaction of PAK4 or JIK with MKK7 and thefunction of either protein, such as kinase activity, is not disturbed,PAK4, JIK and MKK7 can have a different type of protein or peptideligated at the N-terminus or the C-terminus thereof, directly orindirectly via a linker peptide and the like, by means of, for example,genetic engineering techniques. Alternatively, PAK4, JIK and MKK7 can belabeled with a chemical modifier. Preferably, the labeling is employedthat does not result in inhibiting the basic properties of PAK4, JIK andMKK7. Examples of proteins and peptides to be ligated include enzymessuch as glutathione-S-transferase, β-galactosidase, horse radishperoxidase and alkaline phosphatase, tag peptides such as His-tag,Myc-tag, HA-tag, FLAG-tag and Xpress-tag, maltose binding proteins andan Fc fragment of immunoglobulin. Examples of chemical modifiers usedfor labeling include fluorescent substances (e.g., green fluorescentprotein, fluorescein isothiocyanate and phycoerythrin), biotin, andradioactive isotopes. At the time of the labeling, these proteins andpeptides can be ligated alone or in combination. Detection of theseproteins or peptides themselves used for the labeling, or of thefunction thereof, makes it possible to determine, for example, thebinding of PAK4 or JIK to MKK7. Examples of a compound to be screenedinclude compounds derived from chemical libraries and naturalsubstances, as well as compounds obtained by drug design based on theprimary or the three-dimensional structures of PAK4, JIK and MKK7.

A compound that inhibits the binding of PAK4 to MKK7 can be identified,for example, by selecting conditions that allow for the binding of PAK4to MKK7, contacting PAK4 and MKK7 with a test compound under theconditions, employing an assay system that uses a signal and/or a markercapable of detecting the binding of PAK4 to MKK7, and detecting thepresence, the absence, or the change of the signal and/or the marker.For example, when the signal generated by the binding of PAK4 to MKK7 orthe marker of the binding exhibits a change such as a disappearance ordecrease as a result of the contact of PAK4 and MKK7 with a testcompound, it can be determined that the compound inhibits the binding ofPAK4 to MKK7. In such an identification method, a test compound can bebrought into contact with PAK4 and/or MKK7 in advance of carrying outthe binding reaction of PAK4 to MKK7. The term “signal” as used hereinrefers to a substance that can be detected directly based on itsphysical properties or chemical properties. The term “marker” refers toa substance that can be detected indirectly by using its physicalproperties or biological properties as an indicator. For signals,luciferase, green fluorescent protein, radioactive isotopes and the likecan be used; for markers, reporter genes such as the chloramphenicolacetyl transferase gene, or detectable tags such as 6×His-tag can beused. However, all substances that are well-known can be used. Methodsfor detecting these signals and markers are known to those skilled inthe art.

Specifically, a compound that inhibits the binding of PAK4 to MKK7 canbe identified, for example, by carrying out an evaluation after adding atest compound to a common in-vitro binding assay, which is known tothose skilled in the art, comprising fixing either PAK4 or MKK7 on asolid-phase, carrying out a binding reaction using the other one of PAK4or MKK7, which is labeled with a signal, and measuring quantitativelythe signal used for labeling.

Alternatively, a compound that inhibits the phosphorylation of MKK7caused by PAK4 can be identified by selecting conditions that allow forthe phosphorylation of MKK7 by PAK4, contacting PAK4 and MKK7 with atest compound under the conditions, employing an assay system that usesa signal and/or a marker capable of detecting the phosphorylation ofMKK7 by PAK4, and detecting the presence, the absence, or the change ofthe signal and/or the marker. For instance, when the signal resultingfrom the phosphorylation of MKK7 by PAK4 or the marker of thephosphorylation exhibits change, such as a disappearance or decrease asa result of the contact of PAK4 and MKK7 with a test compound, it can bedetermined that the compound inhibits the phosphorylation of MKK7 causedby PAK4. In such an identification method, a test compound can bebrought into contact with PAK4 and/or MKK7 in advance of carrying outthe phosphorylation reaction of MKK7 by PAK4. A protein phosphorylationanalysis and quantitative method for measuring phosphorylated proteincan be carried out using methods well-known in the art. The proteinphosphorylation analysis and quantitative method for measuringphosphorylated protein can be carried out in a simple way, for example,as shown in the examples described below, by bringing PAK4 to react withMKK7 in-vitro in the presence of adenosine triphosphate (ATP) labeledwith a radioactive isotope (³²P), separating the proteins using SDS-PAGEafter the reaction, detecting the bands showing the proteins by way ofstaining, and then, measuring the radioactivity of the bandcorresponding to the phosphorylated MKK7.

Alternatively, a compound that inhibits the binding of PAK4 to MKK7and/or the phosphorylation of MKK7 caused by PAK4 can be identified byusing cells in which PAK4 and MKK7 have been expressed, bringing thecells into contact with a test compound, inhibiting the binding of PAK4to MKK7 or the binding of JIK to MKK7 among the antibodies obtained.Antibodies can be produced, for example, using each protein itself suchas PAK4, JIK and MKK7, or the peptides or the oligopeptides (comprisingthe amino acid sequences of the interaction sites of PAK4 with MKK7 orof JIK with MKK7) as an antigen, by using antibody preparation methodswell-known in the art.

The thus-selected compounds, binding inhibitors and phosphorylationinhibitors can be used as effective ingredients in a medicament based oninhibiting the binding of PAK4 to MKK7, the binding of JIK to MKK7, thephosphorylation of MKK7 by PAK4 or the phosphorylation of MKK7 by JIK,by way of further selection with consideration given to the balancebetween the biological effectiveness and toxicity thereof. Both PAK4 andJIK directly phosphorylate and activate MKK7 after binding to MKK7,which results in the activation of JNK3, followed by the phosphorylationof c-Jun. Therefore, the aforementioned compounds, binding inhibitorsand phosphorylation inhibitors can be used as an effective ingredient ina medicament for a disorder attributable to c-Jun phosphorylation byJNK3, such as a disorder attributable to apoptosis, practically, aneurodegenerative disorder.

The medicament according to the present invention can be a medicamentcontaining an effective dose of at least one member selected from thegroup consisting of the aforementioned compounds, binding inhibitors andphosphorylation inhibitors, but it is preferably a pharmaceuticalcomposition formulated in combination with suitable pharmaceuticalacceptable carriers or vehicles. Examples of a carrier includephysiological saline, buffered physiological saline, dextrose, water,glycerol, ethanol, and a mixture thereof; however, they are not limitedthereto.

Suitable dosage ranges can be determined according to the following:effectiveness of the aforementioned compounds, binding inhibitors andphosphorylation inhibitors; the route of administration; the type of thedisorder; the characteristics of the subject (e.g., body weight, age,symptomatic conditions and whether being taking other medicaments); andthe judgment of the doctor in charge. More specifically, a suitabledosage may fall within the range of, e.g., 0.1 μg to 10 μg per 1 kg ofthe body weight of the subject. However, the dosage may be altered usingcommon conventional experiments for optimization of a dosage that arewell known in the art. The aforementioned dosage can be divided foradministration once to four times a day. Alternatively, periodicadministration once every few days or few weeks can be employed.

In terms of a formulation, the formulation can be selected according toan administration route, and such formulations are well-known to thoseskilled in the art. At the time of formulation, the compositions can beused alone or in combination with other compounds or medicamentsrequired for treatment. For example, an effective ingredient, such as ac-Jun phosphorylation inhibitor or an agent for preventing and/ortreating for a nerve degenerative disorder can be formulated together.

In terms of the mode of administration, it may be either systemicadministration or local administration. A mode of administration that isappropriate for a particular disorder, symptomatic conditions, or otherfactors should be selected. One preferred mode of systemicadministration is injection, e.g., an intravenous injection orintra-arterial injection. Other injection routes, such as subcutaneous,intramuscular, or intraperitoneal injection, may also be used. Anothermode of administration may be oral administration, so long as an entericformulation or a capsule formulation can be suitably formulated. Inaddition, transmucosal administration or percutaneous administration,which comprises using a penetrant such as bile salt, fusidic acid, orother surfactants, may also be used. In a local administration, formssuch as ointments, pastes, or gels, may be used.

Preparation of a pharmaceutical may be carried out by well-knownformulating procedures, where a suitable carrier for formulation can beused in accordance with the mode of administration or the physicalproperties of the effective ingredient therein. Specifically,formulations such as powdered drugs, pills, tablets, capsules, aqueoussolutions, ethanol solutions, liposomal formulations, fat emulsions,clathrates (such as those of cyclodextrin), and the like can be used.

Powdered drugs, pills, capsules, or tablets can be prepared using, forexample, an excipient such as lactose, glucose, sucrose, or mannitol; adisintegrant such as starch or sodium arginate; a lubricant such asmagnesium stearate or talc; a binder such as polyvinylalcohol,hydroxypropyl cellulose, or gelatin; a surfactant such as fatty acidester; and a plasticizer such as glycerin, or the like. For preparationof a tablet or capsule, a solid pharmaceutical acceptable carrier isused.

A suspension can be prepared using water, saccharides such as sucrose,sorbitol, or fructose, glycols such as PEG, and oils.

Injectable solutions can be prepared using a carrier comprising a saltsolution, a glucose solution, or a mixture of a salt solution and aglucose solution.

Inclusion into a liposome formulation may be conducted in the followingmanner: by dissolving the substance of interest in a solvent (e.g.,ethanol) to make a solution, adding a solution of phospholipidsdissolved in an organic solvent (e.g., chloroform), removing the solventby evaporation and adding a phosphate buffer thereto, agitating thesolution and then subjecting it to sonication followed by centrifugationto obtain supernatant, and finally, filtrating the supernatant torecover liposomes.

A fat emulsion can be prepared in the following manner: by mixing thesubstance of interest, an oil ingredient (vegetable oil such as soybeanoil, sesame oil, olive oil, or MCT), an emulsifier (such as aphospholipid), and the like; heating the mixture to make a solution;adding water of a required quantity; and then emulsifying orhomogenizing by use of an emulsifier (a homogenizer, e.g., a highpressure jet type, an ultrasonic type, or the like). The fat emulsionmay be also lyophilized. For conducting lipid-emulsification, anauxiliary emulsifier may be added, and examples thereof include glycerinor saccharides (e.g., glucose, sorbitol, fructose, etc.).

Inclusion into a cyclodextrin formulation may be carried out in thefollowing manner: by dissolving the substance of interest in a solvent(e.g., ethanol); adding a solution of cyclodextrin dissolved in waterunder heating thereto; chilling the solution and filtering theprecipitates; and drying under sterilization. At this time, thecyclodextrin to be used may be appropriately selected from among thosehaving different void sizes (α, β, or γ type) in accordance with thebulkiness of the substance of interest.

The present invention provides a reagent kit that includes at least onemember selected from the group consisting of PAK4, JIK, a polynucleotideencoding PAK4, a polynucleotide encoding JIK, a vector containing thepolynucleotide encoding PAK4 and a vector containing the polynucleotideencoding JIK, along with at least one member selected from the groupconsisting of MKK7, a polynucleotide encoding MKK7 and a vectorcontaining the polynucleotide encoding MKK7. JIK, PAK4 and MKK7 can becontained in cells in which these are expressed by means of geneticengineering techniques, the products of cell-free synthesis systems,chemical synthesis products, or can be obtained from cells or from anybiological samples. These can be subsequently further purified for use.Furthermore, as long as the binding of JIK or PAK4 to MKK7 and thefunction of these proteins, such as kinase activity, is not disturbed,JIK, PAK4 and MKK7 can have a different type of protein or peptideligated at the N-terminus or the C-terminus thereof, directly orindirectly via a linker peptide and the like, by means of, for example,genetic engineering techniques. Alternatively, PAK4, JIK and MKK7 can belabeled with a chemical modifier. Preferably, the labeling is employedthat does not result in inhibiting the basic properties of PAK4, JIK andMKK7. Examples of proteins and peptides to be ligated include enzymessuch as glutathione-S-transferase, P-galactosidase, horse radishperoxidase and alkaline phosphatase, tag peptides such as His-tag,Myc-tag, HA-tag, FLAG-tag and Xpress-tag, maltose binding proteins andan Fc fragment of immunoglobulin. Examples of chemical modifiers usedfor the labeling include fluorescent substances (e.g., green fluorescentprotein, fluorescein isothiocyanate and phycoerythrin), biotin, andradioactive isotopes. At the time of the labeling, these proteins andpeptides can be ligated alone or in combination. A polynucleotideencoding any one of PAK4, JIK and MKK7 can be prepared from human cDNAlibrary using genetic engineering techniques that are well known in theart. A vector containing the polynucleotide encoding any one of PAK4,JIK and MKK7 can be obtained by introducing the polynucleotide into asuitable expression vector DNA, such as a vector derived from abacterial plasmid, using genetic engineering techniques that are wellknown in the art. The reagent kit may contain required substances, suchas a signal and/or marker for detecting the binding of PAK4 or JIK toMKK7, or for detecting the phosphorylation of MKK7 by PAK4 or JIK, abuffer, and a salt. Further, they may contain a stabilizer and/orantiseptic agent. In terms of the preparation thereof, it is sufficientto use a well-known means to prepare each substance.

EXAMPLES

Hereinafter, the present invention may be explained more particularlywith an example; however, the present invention is not limited to thefollowing examples.

Example 1

(In-Silico Search for Proteins Having a Function of Interacting withMKK7)

The prediction of proteins having a function of interacting with MKK7was conducted according to the method set forth in the InternationalPatent Publication No: WO 01/67299. First, the amino acid sequence ofMKK7 was decomposed into oligopeptides having a pre-determined length inorder to search in a database for proteins having the amino acidsequence of each of the oligopeptides, or having homologous amino acidsequences to these amino acid sequences. Next, local alignment wasconducted between the proteins obtained and MKK7 to identify proteinshaving a high local alignment score and that might be capable ofinteracting with MKK7.

As a result of the analysis, it was found that the oligopeptides DIWSLGI(SEQ ID NO: 3) and PPARAR (SEQ ID NO: 4), which have homology to theoligopeptides DVWSLGI (SEQ ID NO: 1) and PPARPR (SEQ ID NO: 2) thatconsist of seven or six amino acid residues derived from MKK7, arepresent in the amino acid sequence of the PAK4. FIG. 1 shows the resultsof local alignment between MKK7 and PAK4. From these results, PAK4 waspredicted to be a protein having a function of interacting with MKK7.

Example 2

(Analysis of the Phosphorylation of MKK7 by PAK4)

In order to experimentally determine the interaction of PAK4 with MKK7,an in-vitro phosphorylation experiment was conducted using the immunecomplex phosphorylation method.

<Materials>

A PAK4 expression plasmid was constructed as follows. First, human PAK4cDNA was obtained from human-brain-derived poly(A)⁺RNA (Clontech) by areverse transcription polymerase chain reaction (RT-PCR), and theninserted into pcDNA3.1(+) (Invitrogen) which is an expression vector foran animal cell expression system. At the time of insertion of the cDNAinto the vector, a FLAG-tag coding sequence or HA-tag coding sequencewas inserted at the 5′ side, and thereby there were constructedrespectively an expression plasmid for an animal cell expression systemcomprising PAK4 ligated with FLAG-tag at its N-terminus(pcDNA-FLAG-PAK4) and comprising PAK4 ligated with HA-tag at itsN-terminus (pcDNA-HA-PAK4).

Buffers comprising the following compositions were used in theexperiments.

Cell lysis buffer: 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM ethylenediamine tetraacetic acid (EDTA), 1 mM ethylene glycol bis-tetraaceticacid (EGTA), 1% Triton X-100, 2.5 mM Na-pyrophosphate, 1 mMβ-glycerophosphate, 1 mM Na₃VO₄, protease inhibitor cocktail (CellSignaling Technology).

Kinase buffer: 25 mM Tris-HCl, pH 7.5, 5 mM β-glycerophosphate, 2 mMdithiothreitol, 0.1 mM Na₃VO₄, 10 mM MgCl₂ (Cell Signaling Technology).

SDS sample buffer: 4% SDS, 125 mM Tris-HCl, pH 6.8, 20% glycerol, 0.01%bromo phenol blue (BPB), 10% β-mercaptoethanol.

<Method>

After HEK293 cells (5.105 cells in a dish with 60 mm diameter) werecultured overnight at 37° C. in 5% CO₂, the cells were transfected with5 μg pcDNA-FLAG-PAK4 by using 15 μl FuGENE6 Transfection Reagent(Roche). The transfection was similarly conducted using pcDNA3.1(+) as anegative control. After culturing for two days, cells were rinsed withice-cold phosphate-buffered physiological saline (−) (PBS (−)),collected and suspended in the 500 μl cell lysis buffer, and leftstanding on ice for 10 minutes. Then, the cells were subjected tocentrifugation at 14,000 rpm for 10 minutes at 4° C. to collect thesupernatant for use as a cell lysate. Next, 500 μl cell lysate was mixedwith 20 μl agarose-conjugated normal mouse IgG (Sigma) by inversion for30 minutes at 4° C., followed by centrifugation to collect thesupernatant. The collected supernatant was mixed with 20 μl anti-FLAG M2affinity gel (Sigma) by inversion for two hours at 4° C. Then, the beadswere collected by centrifugation and washed twice with 500 μl cell lysisbuffer and twice with 500 μl kinase buffer. Next, the beads were mixedwith 25 μl kinase buffer containing 1 μg substrate, 10 μM ATP and 5 μCi[γ-³²P]ATP (3,000Ci/mmol, PerkinElmer) and a phosphorylation reactionwas allowed to proceed for 30 minutes at 30° C. After the reaction, 25μl 2.SDS sample buffer was added. After boiling for five minutes, thesupernatant was separated by SDS-PAGE and phosphorylated proteins weredetected by autoradiography using BAS2000 (Fuji film). As a substrate,unactive GST-MKK7 (Upstate) was used, while GST was used for a negativecontrol.

<Results>

The phosphorylation of GST-MKK7 by PAK4 was observed as shown in FIG. 2.Since such phosphorylation was not observed in the absence of PAK4, itwas revealed that the phosphorylation of GST-MKK7 was notself-phosphorylation, but was caused by PAK4. The phosphorylation wasnot observed when GST was used as a substrate for a negative control.

Example 3

(Analysis of the Binding of PAK4 to MKK7)

In order to experimentally determine the binding of PAK4 to MKK7, abinding test was conducted using theintracellular-co-expression/immuno-co-precipitation method.

<Materials>

For the PAK4 expression plasmid, the plasmid constructed in example 2was used.

An MKK7 expression plasmid was constructed as follows. First, human MKK7cDNA was obtained from human skeletal muscle-derived poly(A)⁺RNA(Clontech) by RT-PCR and then inserted into pcDNA3.1(+) (Invitrogen)which is an expression vector for an animal cell expression system. Atthe time of insertion of the cDNA into the vector, an HA-tag codingsequence was inserted at the 5′ side, and thereby an expression plasmidfor an animal cell expression system of MKK7 ligated with HA-tag at itsN-terminus (pcDNA-HA-MKK7) was constructed.

The buffers used in the experiments were the same in composition asthose described in Example 2.

<Method>

After HEK293T cells (4.10⁵ cells in a dish with 60 mm diameter) werecultured overnight at 37° C. in 5% CO₂, the cells were transfected with2 μg pcDNA-FLAG-PAK4 along with 2 μg pcDNA-HA-MKK7 by using FuGENE6Transfection Reagent (Roche). The transfection was similarly conductedusing pcDNA3.1(+) as a negative control. After culturing for two days,cells were rinsed with ice-cold PBS (−), collected and suspended in the500 μl cell lysis buffer, and left standing on ice for 10 minutes. Then,the cells were subjected to centrifugation at 14,000 rpm for 10 minutesat 4° C. to collect the supernatant for use as a cell lysate. Next, 500μl cell lysate was mixed with 20 μl agarose-conjugated normal mouse IgG(Sigma) by inversion for 30 minutes at 4° C., followed by centrifugationto collect the supernatant. The collected supernatant was mixed with 20μl anti-FLAG M2 affinity gel (Sigma) by inversion overnight at 4° C.Then, the beads were collected by centrifugation, washed three timeswith 500 μl cell lysis buffer and once with 500 μl Tris-bufferedphysiological saline (TBS: 25 mM Tris-HCl, pH 7.5, 150 mM NaCl), andthen mixed with 20 μl 2.SDS sample buffer. After boiling for fiveminutes, the supernatant was separated by 5-20% SDS-PAGE. Then, bindingproteins were detected by western blotting using an anti-HA antibody(Y-11, SantaCruz). An ECL western blotting detection kit (Amershampharmacia biotech) was used for the detection.

<Results>

As a result of the immuno-precipitation of FLAG-PAK4 using an anti-FLAGantibody, HA-MKK7 was co-precipitated therewith when using the cellsco-expressing FLAG-PAK4 and HA-MKK7, as shown in FIG. 3. On the otherhand, HA-MKK7 was not co-precipitated when using the cells notexpressing FLAG-PAK4, which revealed that such co-precipitation resultedfrom the binding of FLAG-PAK4 to HA-MKK7 rather than nonspecific bindingto the agarose beads. These findings revealed that PAK4 binds to MKK7 ina cell.

Example 4

(Activation of the JNK3 Signaling Pathway by PAK4)

In order to experimentally determine the activation of the JNK3signaling pathway by PAK4, an in-vitro JNK3 phosphorylation experimentwas conducted using the immune complex phosphorylation method.

<Materials>

For a PAK4 expression plasmid, the plasmid constructed in example 2 wasused.

A JNK3 expression plasmid was constructed as follows. First, human JNK3cDNA was obtained from a human hippocampal cDNA library by RT-PCR, andthen inserted into pcDNA3.1(+) (Invitrogen) which is an expressionvector for an animal cell expression system. At the time of theinsertion of the cDNA into the vector, a FLAG-tag coding sequence wasinserted at the 5′ side, and thereby an expression plasmid for an animalcell expression system of JNK3 ligated with FLAG-tag at its N-terminus(pcDNA-FLAG-JNK3) was constructed.

Meanwhile, c-Jun (1-79) (a region of c-Jun consisting of its N-terminal79 amino acids that includes the JNK phosphorylation site) was expressedas a fusion protein ligated with GST (glutathione S-transferase) at itsN-terminus (hereinafter, GST-c-Jun (1-79)) in Escherichia coli, and thenpurified with glutathione sepharose 4B (Amersham Pharmacia biotech) foruse.

The solutions used in the experiment were the same in composition asthose described in Example 2.

<Method>

After HEK293 cells (6.10⁵ cells in a dish with 60 mm diameter) werecultured overnight at 37° C. in 5% CO₂, the cells were transfected withpcDNA-FLAG-JNK3 (2 μg) and pcDNA-HA-PAK4 (0, 0.1, 0.5 or 2 μg) by using12 μl FuGENE6 Transfection Reagent (Roche). The total amount of DNA wasadjusted to 4 μg each using pcDNA 3.1(+). After culturing for two days,the cells were rinsed with ice-cold PBS (−), collected and suspended inthe 500 μl cell lysis buffer, and left standing on ice for 10 minutes.Then, the cells were subjected to centrifugation at 14,000 rpm for 10minutes at 4° C. to collect the supernatant for use as a cell lysate.Next, 500 μl cell lysate was mixed with 20 μl agarose-conjugated normalmouse IgG (Sigma) by inversion for 30 minutes at 4° C., followed bycentrifugation to collect the supernatant. The collected supernatant wasmixed with 20 μl anti-FLAG M2 affinity gel (Sigma) by inversion fortwo-hours at 4° C. Then, the beads were collected by centrifugation andwashed twice with 500 μl cell lysis buffer and twice with 500 μl kinasebuffer. Next, the beads were mixed with 25 μl kinase buffer containing 2μg GST-c-Jun (1-79) as a substrate, 10 μM ATP and 5 μCi [γ-³²P]ATP(3,000 Ci/mmol, PerkinElmer) and the phosphorylation reaction wasallowed to proceed for 30 minutes at 30° C. After the reaction, 25 μl2.SDS sample buffer was added. After boiling for five minutes at 100°C., the supernatant was separated by SDS-PAGE and the phosphorylatedGST-c-Jun(1-79) was detected by autoradiography using BAS2000 (Fujifilm). The expression of each protein was detected by western blottingusing anti-FLAG M2 monoclonal antibody (Sigma) or anti-HA antibody(Y-11, SantaCruz).

<Results>

Phosphorylated c-Jun increased depending on the amount of expressedHA-PAK4, in the kinase assay using a lysate of cells co-expressingHA-PAK4 and FLAG-JNK3, as shown in FIG. 4. Specifically, it was revealedthat the expression of HA-PAK4 increases the c-Jun phosphorylationactivity of JNK3. Since the amount of expressed FLAG-JNK3 did not show alarge variation, the increase in JNK3 activity can be considered toresult from the expression of HA-PAK4.

The results obtained in the examples 2, 3 and 4 revealed that PAK4 bindsto MKK7 and directly phosphorylates MKK7, which results in theactivation of JNK3 followed by the phosphorylation of c-Jun, that is tosay, the activation of JNK3 signaling pathway. Example 5

(In-Silico Search for Proteins Having a Function of Interacting withMKK7)

The prediction of proteins having a function of interacting with MKK7was conducted in the same way as in Example 1. As a result, it was foundthat the oligopeptides WSLGIT (SEQ ID NO: 7) and LENKLK (SEQ ID NO: 8),which have homology to the oligopeptides WSLGIS (SEQ ID NO: 5) andLEAKLK (SEQ ID NO: 6) that consist of six amino acid residues derivedfrom MKK7, are present in the amino acid sequence of the JIK. FIG. 5shows the results of local alignment between MKK7 and JIK. From theseresults, JIK was predicted to be a protein having a function ofinteracting with MKK7.

Example 6

(Analysis of the Phosphorylation of MKK7 by JIK)

In order to experimentally determine the phosphorylation of MKK7 by JIK,an in-vitro phosphorylation experiment was conducted using the immunecomplex phosphorylation method.

<Materials>

A JIK expression plasmid was constructed as follows. First, human JIKcDNA was obtained from human kidney-derived poly(A)⁺RNA (Clontech) byRT-PCR, and then inserted into pcDNA3.1(+) (Invitrogen) which is anexpression vector for an animal cell expression system. At the time ofinsertion of the cDNA into the vector, an HA-tag coding sequence wasinserted at the 5′ side, and thereby an expression plasmid for an animalcell expression system of JIK ligated with HA-tag at its N-terminus(pcDNA-HA- JIK) was constructed. The coding amino acid sequence ofcloned JIK cDNA is the same as that of the accession number XP_(—)045006(registered gene name: JIK) disclosed in the NCBI (National Center forBiotechnology Information) protein database.

The buffers used in the experiment were the same in composition as thosedescribed in Example 2.

<Method>

After HEK293T cells (4.10⁵ cells in a dish with 60 mm diameter) werecultured overnight at 37° C. in 5% CO₂, the cells were transfected with5 μg pcDNA-HA-JIK by using 15 μl FuGENE6 Transfection Reagent (Roche).The transfection was similarly conducted using pcDNA3.1(+) as a negativecontrol. After culturing for two days, cells were rinsed with ice-coldPBS (−), collected and suspended in the 500 μl cell lysis buffer, andleft standing on ice for 10 minutes. Then, the cells were subjected tocentrifugation at 14,000 rpm for 10 minutes at 4° C. to collect thesupernatant for use as a cell lysate. Next, 500 μl cell lysate was mixedwith 20 μl agarose-conjugated normal mouse IgG (Sigma) by inversion for30 minutes at 4° C., followed by centrifugation to collect thesupernatant. The collected supernatant was mixed with 20 μl anti-HAaffinity matrix (Roche) by inversion for two hours at 4° C. Then, thebeads were collected by centrifugation and washed twice with 500 μl celllysis buffer and twice with 500 μl kinase buffer. Next, the beads weremixed with 25 μl kinase buffer containing 1 μg substrate, 10 μM ATP and5 μCi [γ-³²P]ATP (3,000 Ci/mmol, PerkinElmer) and the phosphorylationreaction was allowed to proceed for 30 minutes at 30° C. After thereaction, 25 μl 2.SDS sample buffer was added. After boiling for fiveminutes, the supernatant was separated by SDS-PAGE and phosphorylatedproteins were detected by autoradiography using BAS2000 (Fuji film). Asa substrate, inactive GST-MKK7 (Upstate) was used, while GST was usedfor a negative control.

<Results>

The phosphorylation of GST-MKK7 by JIK was observed as shown in FIG. 6.Since such phosphorylation was not observed in the absence of JIK, itwas revealed that the phosphorylation of GST-MKK7 was notself-phosphorylation, but was caused by JIK. The phosphorylation was notobserved when GST was used as a substrate for a negative control.

Example 7

(Analysis of the Binding of JIK to MKK7)

In order to experimentally determine the binding of JIK to MKK7, abinding test was conducted using theintracellular-co-expression/immuno-co-precipitation method.

<Materials>

For a JIK expression plasmid, the plasmid constructed in example 6 wasused.

An MKK7 expression plasmid was constructed as follows. First, human MKK7cDNA was obtained from human skeletal muscle-derived poly(A)⁺RNA(Clontech) by RT-PCR, and then inserted into pcDNA3.1(+) (Invitrogen)which is an expression vector for an animal cell expression system. Atthe time of insertion of the cDNA into the vector, FLAG-tag codingsequence was inserted at the 5′ side, and thereby an expression plasmidfor an animal cell expression system of MKK7 ligated with FLAG-tag atits N-terminus (pcDNA-FLAG-MKK7) was constructed.

The buffers used in this test were the same in composition as thosedescribed in Example 2.

<Method>

After HEK293T cells (4.10⁵ cells in a dish with 60 mm diameter) werecultured overnight at 37° C. in 5% CO₂, the cells were transfected with2 μg pcDNA-HA-JIK along with 2 μg pcDNA-FLAG-MKK7 by using FuGENE6Transfection Reagent (Roche). The transfection was similarly conductedusing pcDNA3.1 (+) as a negative control. After culturing for two days,cells were rinsed with ice-cold PBS (−), collected and suspended in the500 μl cell lysis buffer, and left standing on ice for 10 minutes. Then,the cells were subjected to centrifugation at 14,000 rpm for 10 minutesat 4° C. to collect the supernatant for use as a cell lysate. Next, 500μl cell lysate was mixed with 20 μl agarose-conjugated normal mouse IgG(Sigma) by inversion for 30 minutes at 4° C., followed by centrifugationto collect the supernatant. The collected supernatant was mixed with 20μl anti-HA affinity matrix (Roche) by inversion overnight at 4° C. Then,the beads were collected by centrifugation, washed three times with 500μl cell lysis buffer and once with 500 μl TBS (25 mM Tris-HCl, pH 7.5,150 mM NaCl), and then mixed with 20 μl 2.SDS sample buffer. Afterboiling for five minutes, the supernatant was separated by 5-20%SDS-PAGE. Then, binding proteins were detected by western blotting usingan anti-FLAG M2 antibody (Sigma). An ECL western blotting detection kitwas used for the detection.

<Results>

As a result of the immuno-precipitation of HA-JIK using the anti-HAantibody, FLAG-MKK7 was co-precipitated therewith when using the cellsco-expressing HA-JIK and FLAG-MKK7, as shown in FIG. 7. On the otherhand, FLAG-MKK7 was not co-precipitated when using the cells notexpressing HA-JIK, which revealed that such co-precipitation resultedfrom the binding of HA-JIK to FLAG-MKK7 rather than nonspecific bindingto the agarose beads. These findings revealed that JIK binds to MKK7 ina cell.

Example 8

(Activation of the JNK3 Signaling Pathway by JIK)

In order to experimentally determine the activation of the JNK3signaling pathway by JIK, an in-vitro JNK3 phosphorylation experimentwas conducted using the immune complex phosphorylation method.

<Materials>

For a JNK3 expression plasmid, the plasmid constructed in example 4 wasused.

For a JIK expression plasmid, the plasmid constructed in example 6 wasused.

GST-c-Jun (1-79) was prepared in the same manner as in example 4.

The buffers used in this test were the same in composition as thosedescribed in Example 2.

<Method>

After HEK293T cells (4.10⁵ cells in a dish with 60 mm diameter) werecultured overnight at 37° C. in 5% CO₂, the cells were transfected with2 μg pcDNA-HA-JIK and 2 μg pcDNA-FLAG-JNK3 by using FuGENE6 TransfectionReagent (Roche). The transfection was similarly conducted usingpcDNA3.1(+) as a negative control. After culturing for two days, cellswere rinsed with ice-cold PBS (−), collected and suspended in the 500 μlcell lysis buffer, and left standing on ice for 10 minutes.

Then, the cells were subjected to centrifugation at 14,000 rpm for 10minutes at 4° C. to collect the supernatant for use as a cell lysate.Next, 500 μl cell lysate was mixed with 20 μl agarose-conjugated normalmouse IgG (Sigma) by inversion for 30 minutes at 4° C., followed bycentrifugation to collect the supernatant. The collected supernatant wasmixed with 20 μl anti-FLAG M2 affinity gel (Sigma) by inversion for twohours at 4° C. Then, the beads were collected by centrifugation andwashed twice with 500 μl cell lysis buffer and twice with 500 μl kinasebuffer. Next, the beads were mixed with 25 μl kinase buffer containing 2μg GST-c-Jun (1-79) as a substrate, 10 μM ATP and 5 μCi [γ-³²P]ATP(3,000 Ci/mmol, PerkinElmer) and the phosphorylation reaction wasallowed to proceed for 30 minutes at 30° C. After the reaction, 25 μl2.SDS sample buffer was added. After boiling for five minutes at 100°C., the supernatant was separated by SDS-PAGE and the phosphorylatedGST-c-Jun(1-79) was detected by autoradiography using BAS2000 (Fujifilm).

<Results>

Phosphorylated c-Jun increased in the kinase assay using a lysate ofcells co-expressing HA-JIK and FLAG-JNK3 in comparison with the kinaseassay using a lysate of cells expressing FLAG-JNK3 only, as shown inFIG. 8. Specifically, it was revealed that HA-JIK increases the c-Junphosphorylation activity of JNK3.

The results obtained in the examples 6, 7 and 8 revealed that JIK bindsto MKK7 and directly phosphorylates MKK7, which results in theactivation of JNK3 followed by the phosphorylation of c-Jun, that is tosay, the activation of JNK3 signaling pathway.

INDUSTRIAL APPLICABILITY

In the present invention, it was discovered for the first time that bothPAK and JIK bind to MKK7, and that both PAK and JIK directlyphosphorylate MKK7, and that as a result, the JNK3 signaling pathway isactivated.

Although it has been already known that PAK4 activates the JNK signalingpathway, the mechanism thereof was unascertained. PAK4 is activated bycdc42. The activation of cdc42 gives rise to nerve cell death via theactivation of the JNK signaling pathway. Therefore, the MKK7phosphorylation by PAK4 is presumably involved in nerve cell deathcaused by the activation of the JNK3 signaling pathway.

Although it has been already reported that JIK is involved in the JNKactivation caused by ER stress, the mechanism thereof was unascertained.ER stress gives rise to nerve cell death via the activation of the JNKsignaling pathway. Therefore, the MKK7 phosphorylation by JIK ispresumably involved in nerve cell death caused by the activation of theJNK3 signaling pathway.

Consequently, it is possible to inhibit the phosphorylation of c-Junresulting from the activation of JNK3 signaling pathway by inhibitingthe binding of PAK4 or JIK to MKK7, or the phosphorylation of MKK7 byPAK4 or JIK, and further to inhibit nerve cell death.

Since both PAK4 and JIK directly phosphorylate MKK7, PAK4 and JIKpresumably phosphorylate MKK7 through different pathways, and thereforeare both involved in the activation of the JNK3 signaling pathway.Therefore, to inhibit both the activation of MKK7 caused by PAK4 andthat caused by JIK may provide a more enhanced effect in inhibiting thephosphorylation of c-Jun than to inhibit only the activation of MKK7caused by either PAK4 or JIK.

The present invention is extremely useful for prevention and treatmentof disorders caused by the activation of the JNK3 signaling pathway,such as disorders attributable to nerve cell death, more practically,neurodegenerative disorders; and also for the study of neurodegenerativedisorders and the JNK signaling mechanism.

Free Text in Sequence Listing

-   SEQ ID NO: 1: Partial sequence of MKK7, which is highly homologous    to that (SEQ ID NO:3) of PAK4-   SEQ ID NO: 2: Partial sequence of MKK7, which is highly homologous    to that (SEQ ID NO:4) of PAK4-   SEQ ID NO: 3: Partial sequence of PAK4, which is highly homologous    to that (SEQ ID NO:1) of MKK7-   SEQ ID NO: 4: Partial sequence of PAK4, which is highly homologous    to that (SEQ ID NO:2) of MKK7-   SEQ ID NO: 5: Partial sequence of MKK7, which is highly homologous    to that (SEQ ID NO:7) of JIK-   SEQ ID NO: 6: Partial sequence of MKK7, which is highly homologous    to that (SEQ ID NO:8) of JIK-   SEQ ID NO: 7: Partial sequence of JIK, which is highly homologous to    that (SEQ ID NO:5) of MKK7-   SEQ ID NO: 8: Partial sequence of JIK, which is highly homologous to    that (SEQ ID NO:6) of MKK7-   SEQ ID NO: 9: Partial sequence of MKK7, showing a high score in the    local alignment between MKK7 and PAK4-   SEQ ID NO: 10: Partial sequence of PAK4, showing a high score in the    local alignment between MKK7 and PAK4-   SEQ ID NO: 11: Partial sequence identical in the sequences of MKK7,    PAK4 and JIK-   SEQ ID NO: 12: Partial sequence of MKK7, showing a high score in the    local alignment between MKK7 and PAK4-   SEQ ID NO: 13: Partial sequence of PAK4, showing a high score in the    local alignment between MKK7 and PAK4-   SEQ ID NO: 14: Partial sequence of MKK7, showing a high score in the    local alignment between MKK7 and PAK4-   SEQ ID NO: 15: Partial sequence of PAK4, showing a high score in the    local alignment between MKK7 and PAK4-   SEQ ID NO: 16: Partial sequence identical in the sequences of MKK7    and PAK4-   SEQ ID NO: 17: Partial sequence of MKK7, showing a high score in the    local alignment between MKK7 and PAK4-   SEQ ID NO: 18: Partial sequence of PAK4, showing a high score in the    local alignment between MKK7 and PAK4-   SEQ ID NO: 19: Partial sequence of MKK7, showing a high score in the    local alignment between MKK7 and PAK4-   SEQ ID NO: 20: Partial sequence of PAK4, showing a high score in the    local alignment between MKK7 and PAK4-   SEQ ID NO: 21: Partial sequence of MKK7, showing a high score in the    local alignment between MKK7 and PAK4-   SEQ ID NO: 22: Partial sequence of PAK4, showing a high score in the    local alignment between MKK7 and PAK4-   SEQ ID NO: 23: Partial sequence of MKK7, showing a high score in the    local alignment between MKK7 and PAK4-   SEQ ID NO: 24: Partial sequence of PAK4, showing a high score in the    local alignment between MKK7 and PAK4-   SEQ ID NO: 25: Partial sequence of MKK7, showing a high score in the    local alignment between MKK7 and PAK4-   SEQ ID NO: 26: Partial sequence of PAK4, showing a high score in the    local alignment between MKK7 and PAK4-   SEQ ID NO: 27: Partial sequence of MKK7, showing a high score in the    local alignment between MKK7 and PAK4-   SEQ ID NO: 28: Partial sequence of PAK4, showing a high score in the    local alignment between MKK7 and PAK4-   SEQ ID NO: 29: Partial sequence of MKK7, showing a high score in the    local alignment between MKK7 and JIK-   SEQ ID NO: 30: Partial sequence of JIK, showing a high score in the    local alignment between MKK7 and JIK-   SEQ ID NO: 31: Partial sequence of MKK7, showing a high score in the    local alignment between MKK7 and JIK-   SEQ ID NO: 32: Partial sequence of JIK, showing a high score in the    local alignment between MKK7 and JIK-   SEQ ID NO: 33: Partial sequence of MKK7, showing a high score in the    local alignment between MKK7 and JIK-   SEQ ID NO: 34: Partial sequence of JIK, showing a high score in the    local alignment between MKK7 and JIK-   SEQ ID NO: 35: Partial sequence of MKK7, showing a high score in the    local alignment between MKK7 and JIK-   SEQ ID NO: 36: Partial sequence of JIK, showing a high score in the    local alignment between MKK7 and JIK-   SEQ ID NO: 37: Partial sequence of MKK7, showing a high score in the    local alignment between MKK7 and JIK-   SEQ ID NO: 38: Partial sequence of JIK, showing a high score in the    local alignment between MKK7 and JIK-   SEQ ID NO: 39: Partial sequence of MKK7, showing a high score in the    local alignment between MKK7 and JIK-   SEQ ID NO: 40: Partial sequence of JIK, showing a high score in the    local alignment between MKK7 and JIK-   SEQ ID NO: 41: Partial sequence of MKK7, showing a high score in the    local alignment between MKK7 and JIK-   SEQ ID NO: 42: Partial sequence of JIK, showing a high score in the    local alignment between MKK7 and JIK-   SEQ ID NO: 43: Partial sequence of MKK7, showing a high score in the    local alignment between MKK7 and JIK-   SEQ ID NO: 44: Partial sequence of JIK, showing a high score in the    local alignment between MKK7 and JIK-   SEQ ID NO: 45: Partial sequence of MKK7, showing a high score in the    local alignment between MKK7 and JIK-   SEQ ID NO: 46: Partial sequence of JIK, showing a high score in the    local alignment between MKK7 and JIK

1-7. (canceled)
 8. A method for identifying a compound that inhibits thebinding of JNK/SAPK-inhibitory kinase (JIK) to MAP kinase kinase 7(MKK7), comprising contacting JIK and/or MKK7 with a test compound underconditions that allow the binding of JIK to MKK7; and determiningwhether the test compound inhibits the binding of JIK to MKK7, bydetecting the presence, absence or change of a signal generated by thebinding of JIK to MKK7. 9-28. (canceled)